Inbred corn line LLM02BM

ABSTRACT

An inbred corn line, designated LLM02BM, the plants and seeds of the inbred corn line LLM02BM, methods for producing a corn plant, either inbred or hybrid, produced by crossing the inbred corn line LLM02BM with itself or with another corn plant, and hybrid corn seeds and plants produced by crossing the inbred line LLM02BM with another corn line or plant and to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic corn plants produced by that method. This invention also relates to inbred corn lines derived from inbred corn line LLM02BM, to methods for producing other inbred corn lines derived from inbred corn line LLM02BM and to the inbred corn lines derived by the use of those methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims a priority based on provisional application61/662,513 which was filed in the U.S. Patent and Trademark Office onJun. 21, 2012, the entire disclosure of which is hereby incorporated byreference.

FIELD OF THE INVENTION

This invention is in the field of corn breeding. In particular, theinvention relates to an inbred corn line designated LLM02BM thatincludes plants and seeds of inbred corn line LLM02BM. Methods forproducing corn plants, such as inbred corn plants, hybrid corn plants,or other corn plants, as by crossing inbred corn line LLM02BM withitself or any different corn plant are an integral part of thisinvention as are the resultant corn plants including the plant parts andseeds. This invention further relates to methods for producingLLM02BM-derived corn plants, to methods for producing male sterileLLM02BM corn plants and to methods for regenerating such plants fromtissue cultures of regenerable cells as well as the plants obtainedtherefrom. Methods for producing a corn plant containing in its geneticmaterial one or more transgenes and to the transgenic corn plantsproduced by that method are also a part of this invention.

BACKGROUND OF THE INVENTION

Corn (Zea mays L.) is the most important and abundant crop produced inthe United States. Corn is used as human food, livestock feed, and asraw material in industry. The food uses of corn include kernels forhuman consumption, dry milling products such as grits, meal and flour,and wet milling products such as corn starch, corn syrups, and dextrose.Corn oil recovered from corn germ is a by-product of both dry and wetmilling industries. Both grain and non-grain portions of corn plants areused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Corn is used to produce ethanol while corn starch and flour are used inthe paper and textile industries. Corn is also used in adhesives,building materials, foundry binders, laundry starches, explosives,oil-well muds, and other mining applications. Plant parts other than thegrain of corn are also used in industry; for example, stalks and husksare made into paper and wallboard and cobs are used for fuel and to makecharcoal.

The goal of a corn breeder is to improve a corn plant's performance andtherefore, its economic value by combining various desirable traits intoa single plant. Improved performance is manifested in many ways. Higheryields of corn plants contribute to a more abundant food supply, a moreprofitable agriculture and a lower cost of food products for theconsumer. Improved quality makes corn kernels more nutritious. Improvedplant health increases the yield and quality of the plant and reducesthe need for application of protective chemicals. Adapting corn plantsto a wider range of production areas achieves improved yield andvegetative growth Improved plant uniformity enhances the farmer'sability to mechanically harvest corn.

Corn is a monoecious plant, i.e., corn has imperfect flowers—male,pollen-producing flowers and separate female, pollen receiving flowerson the same plant. The male flowers are located at the top of the plantin the tassel, and the female flowers are located about midway up thestalk in the ear shoot. Each male flower has three anthers and eachfemale flower includes a husk that envelops the cob and silks thatemerge from the end of the cob and husks. Pollination is consummated bytransfer of pollen from the tassels of the male flower to the silks ofthe female flowers.

Because corn has separate male and female flowers, corn breedingtechniques take advantage of the plant's ability to be bred by bothself-pollination and cross-pollination. Self-pollination occurs whenpollen from the male flower is transferred to a female flower on thesame plant. Cross-pollination occurs when pollen from the male flower istransferred to a female flower on a different plant.

A plant is sib-pollinated (a type of cross-pollination) when individualswithin the same family or line are used for pollination (i.e. pollenfrom a family member plant is transferred to the silks of another familymember plant). Self-pollination and sib-pollination techniques aretraditional forms of inbreeding used to develop new inbred corn linesbut other techniques exist to accomplish inbreeding. New inbred cornlines are developed by inbreeding heterozygous plants and practicingselection for superior plants for several generations untilsubstantially homozygous plants are obtained. During the inbreedingprocess with corn, the vigor of the lines decreases and after asufficient amount of inbreeding, additional inbreeding merely serves toincrease seed of the developed inbred. Inbred corn lines are typicallydeveloped for use in the production of hybrid corn lines.

Natural, or open pollination, occurs in corn when wind blows pollen fromthe tassels to the silks that protrude from the tops of the ear shootand may include both self- and cross-pollination. Vigor is restored whentwo different inbred lines are cross-pollinated to produce the firstgeneration (F₁) progeny. A cross between two defined homozygous inbredcorn plants always produces a uniform population of heterozygous hybridcorn plants and such hybrid corn plants are capable of being generatedindefinitely from the corresponding inbred seed supply.

When two different, unrelated inbred corn parent plants are crossed toproduce an F₁ hybrid, one inbred parent is designated as the male, orpollen parent, and the other inbred parent is designated as the female,or seed parent. Because corn plants are monoecious, hybrid seedproduction requires elimination or inactivation of pollen produced bythe female parent to render the female parent plant male sterile. Thisserves to prevent the inbred corn plant designated as the female fromself-pollinating. Different options exist for controlling male fertilityin corn plants such as manual or mechanical emasculation (ordetasseling), genetic male sterility, and application of gametocides.Incomplete removal or inactivation of the pollen in the female parentplant provides the potential for inbreeding which results in theunwanted production of self-pollinated or sib-pollinated seed.Typically, this seed is unintentionally harvested and packaged withhybrid seed.

The development of new inbred corn plants and hybrid corn plants is aslow, costly interrelated process that requires the expertise ofbreeders and many other specialists. The development of new hybrid cornvarieties in a corn plant breeding program involves numerous steps,including: (1) selection of parent corn plants (germplasm) for initialbreeding crosses; (2) inbreeding of the selected plants from thebreeding crosses for several generations to produce a series of inbredlines, which individually breed true and are highly uniform; and, (3)crossing a selected inbred line with an unrelated line to produce the F₁hybrid progeny having restored vigor.

Inbred corn plants and other sources of corn germplasm are thefoundation material for all corn breeding programs. Despite theexistence and availability of numerous inbred corn lines and othersource germplasm, a continuing need still exists for the development ofimproved germplasm because existing inbred parent corn lines lose theircommercial competitiveness over time. To protect and to enhance yieldproduction, trait technologies and seed treatment options provideadditional crop plan flexibility and cost effective control againstinsects, weeds and diseases, thereby further enhancing the potential ofhybrids with LLM02BM as a parent.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions of PlantCharacteristics

In the description and examples that follow, a number of terms are used.To provide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

Anther Color: Recorded at the time of pollen shed when anthers areactively dehiscing pollen as a standard color name [Light Green (1),Green-Yellow (5), Pale Yellow (6), Yellow (7), Salmon (9), Pink (11),Cherry Red (13), Purple (17), Tan (22)] and Munsell color code.

Anthocyanin in Brace Roots: Relative rating of the expression ofanthocyanin in the brace roots (none, faint, 50% purpling/moderate, anddark) recorded two weeks after flowering.

Biomass: Total amount of above-ground plant material harvested, measuredin tons per acre adjusted for 65% moisture.

Cob Color: Recorded as a standard color name [Pink-Orange (10), Pink(11), Light Red (12), Cherry Red (13), Red (14), White (19)] and Munsellcolor code.

Crude protein: Available and unavailable protein; measured as totalnitrogen multiplied by 6.25 and as a percentage of total dry matter.

Ears Per Stalk: Number of ears with seed set on each plant.

Ear Height: Distance measured in centimeters from the ground to thehighest placed ear node (attachment point of the ear shank) of theuppermost developed ear on the stalk.

Ear Leaves: One or more distinct ear leaves on ear husks at floweringrepresented as present or absent. This may be environmentallyinfluenced, and is recorded as present when ear leaves are present insufficient size (>0.25 inches) and on several plants in the middle ofthe row.

Ear Length: Length of an unhusked ear from the butt to the tip; measuredin centimeters.

Ear-Node Leaf Length: Length of the ear-node leaf; measured incentimeters.

Ear-Node Leaf Width: Width of the ear-node leaf at its widest point;measured in centimeters.

Ear Taper: Relative taper of the unshelled ear; rated as Slight ornearly straight, Average, or Extreme or conical.

Endosperm Type: Region of the kernel between the germ and the seed coat;rated as sweet, extra sweet (sh2), normal starch, high amylase starch,waxy, high protein, high lysine, supersweet (se), high oil andother-specify.

Environments: Number of different geographic locations where two hybridsare grown together and in the same experiment.

50% Pollen (GDU from Planting): Number of GDUs after planting when 50%of the plants are shedding pollen.

50% Silk (GDU from Planting): Number of GDUs after emergence when 50% ofthe plants have extruded silk.

GDU (Growing degree units): Number of heat units accumulated over timecalculated using the Barger Method, wherein the heat units for each24-hour period are: [(Max. temp+Min. temp)/2]−50, with 86° F. thehighest maximum temperature used and 50° F. the lowest minimumtemperature used.

Genotype: Genetic constitution of a plant, plant part, or cell.

Glume Band: Recorded as absent or present.

Glume Color: Color of the glume after exposure to sunlight and justbefore extruding anthers; recorded as a standard color name [Light Green(1), Medium Green (2), Dark Green (3), Very Dark Green (4), Green-Yellow(5), Salmon (9), Pink (11), Cherry Red (13), Red (14), Pale Purple (16)]and Munsell color code.

Hard Endosperm Color: Color of the region of the endosperm between thefloury endosperm and the aleurone layer in yellow dent corn; recorded asa standard color name [Pale Yellow (6), Yellow (7), Yellow-Orange (8),Orange (9), Purple (17), White (19), Tan (22)] and a Munsell color code.

Kernel Crown Color: Color of the portion of the kernel distal to the tipcap; recorded as a standard color name [Pale Yellow (6), Yellow (7),Yellow-Orange (8), Orange (9), White(19)] and Munsell color code.

Kernel Row Alignment: Alignment of kernels scored as straight, slightlycurved, or spiral as determined by standing the unshelled ear on itsbase and looking down at the tip.

Kernel Row Type: Presence (Distinct) or absence (Indistinct) of definedkernel rows.

Kernel Type: Two of the common types for field corn are Flint and Dent;where Flint has mostly hard, glassy endosperm with smooth, hard seedcoats, and Dent has soft cores of starch that cause the end of thekernels to collapse or dent during drying.

Lateral Tassel Branches: Number of primary lateral tassel branches thatoriginate from the central spike.

Leaf Angle: Horizontal (less than 30 degrees), Semi-Erect (30 to 60degrees), Erect (greater than 60 degrees); measured on the upper half ofthe plant as the angle between a line perpendicular to the stalk and theunderside of a leaf at its base.

Leaf Color: Color of the leaf; recorded as standard color name [LightGreen (1), Medium Green (2), Dark Green (3), Very Dark Green (4)] andMunsell color code.

Leaves Above Ear: Number of leaves above the ear leaf.

Munsell Color Code: A system that describes colors based on threecharacteristics: hue, value (lightness), and chroma (color purity).

NDFD (Neutral Detergent Fiber Digestibility): Percentage of neutraldetergent fiber that is digestible; determined in vitro by incubating aground feed sample in live rumen fluid and measuring its disappearanceto simulate the amount and rate of digestion that would occur in therumen.

Number of Kernel Rows: Total number of kernel rows on the ear. If therows are indistinct, then this value is an average number of kernelslocated around the circumference of the ear at the mid-point of itslength.

Phenotype: Physical or external appearance of a plant or plant part.

Plant Height: Plant height in centimeters from the ground to the tip ofthe tassel.

Plots: Number of replicated plantings of a single hybrid that areharvested as data points for analyses in the same experiment.

Population: Number of seeds planted on a per acre basis.

Root Lodging: Percentage of corn plants that lean from the vertical axisat an approximate 30° angle or greater just before anthesis.

Silage moisture: Percentage moisture content of biomass measured atharvest.

Silk Color: Color of the silk three days after its emergence; recordedas standard color name [Light Green (1), Green-Yellow (5), Pale Yellow(6), Yellow (7), Salmon (9), Pink-Orange (10), Pink (11), Cherry Red(13), Purple (17), Tan (22)] and Munsell color code.

Stalk Lodging: Percentage of plants that have broken over at or belowthe top ear node at harvest.

Standard Color Names: Color names include Light Green (1), Medium Green(2), Dark Green (3), Very Dark Green (4), Green-Yellow (5), Pale Yellow(6), Yellow (7), Yellow-Orange (8), Salmon (9), Pink-Orange (10), Pink(11), Light Red (12), Cherry Red (13), Red (14), Red and White (15),Pale Purple (16), Purple (17), Colorless (18), White (19), White Capped(20), Buff (21), Tan (22), Brown (23), Bronze (24), Variegated (25), andOther (26).

Starch: Amount of starch carbohydrates; measured by near-infrared (NIR)spectroscopy as a percentage of dry silage matter.

Tassel Type: The tassel branch shape recorded as erect or spreading. Theangle of the base of each tassel branch is used to indicate thedirection of the branches. Erect longer or lighter tassels that droopover on the tip are classified as erect.

Tillers: Branches that develop from axillary buds at the base of a cornplant; they are morphologically identical to the main stalk and capableof forming their own root system, nodes, internodes, leaves, ears, andtassels. Recorded as present or absent.

Total tract dry matter degradability (TDMD): A compilation of all fiberfractions along the whole tract of the dairy animal; measured as sum ofruminal in situ and post-ruminal in vitro degradabilities as apercentage of dry matter.

II. Inbred Corn Line LLM02BM

A. Inbred Corn Plant LLM02BM

In accordance with one aspect of the present invention, provided is anew yellow dent inbred corn seed and plants thereof designated LLM02BM.The present invention further relates to a method for producing inbredcorn seeds that includes, but is not limited to, the steps of plantingseed of inbred corn LLM02BM in proximity to itself or to different seedfrom a same family or line, growing the resulting corn plants underself-pollinating or sib-pollinating conditions with adequate isolation,and harvesting resultant seed obtained from such inbred plants usingtechniques standard in the agricultural arts such as would be necessaryto bulk-up seed such as for hybrid production. The present inventionalso relates to inbred seed produced by such a method.

In any cross between inbred corn plant LLM02BM and another inbred cornplant, LLM02BM may be designated as the male (pollen parent) or thefemale (seed parent). Optionally, the seed of inbred corn line LLM02BMmay be pre-treated to increase resistance of the seed and/or seedlingsto stressed conditions, and further, the corn plants or surrounding soilmay be treated with one or more agricultural chemicals before harvest.Such agricultural chemicals may include herbicides, insecticides,pesticides and the like. The present invention also relates to a cornplant that expresses substantially all of the physiological andmorphological characteristics of inbred corn plant LLM02BM and to asubstantially homogenous population of corn plants having all thephysiological and morphological characteristics of inbred corn plantLLM02BM. Any corn plants produced from inbred corn plant LLM02BM arecontemplated by the present invention and are, therefore, within thescope of this invention. A description of physiological andmorphological characteristics of corn plant LLM02BM is presented inTable 1.

TABLE 1 Physiological and Morphological Characteristics of LLM02BMCharacteristic Value STALK Plant Height (cm) 191 Ear Height (cm) 78Anthocyanin in Brace Roots None (1) (None, Light, Moderate, Dark)Tillers (Present, Absent) Absent LEAF Leaf Angle (Horizontal,Semi-Erect, Erect) Semi-Erect Leaf Color (standard) Medium Green (2)Leaf Color (Munsell) 5 GY 5/6 Leaves Above Ear (count) 6 Ear-Node LeafWidth (cm) 9 Ear-Node Leaf Length (cm) 77 TASSEL 50% Pollen (GDU fromPlanting) 1586 Tassel Type (Spreading, Erect) Erect Lateral TasselBranches (count) 8 Anther Color (standard) Yellow (7) Anther Color(Munsell) 2.5 Y 8/10 Glume Band (Absent or Present) Absent Glume Color(standard) Medium Green (2) Glume Color (Munsell) 5 GY 5/8 EAR 50% Silk(GDU from Planting) 1651 Silk Color (standard) Bronze (24) Silk Color(Munsell) 5 YR 6/6 Ear Leaves (Presnt, Absent) Absent Ears Per Stalk(count) 1 Ear Length (cm) 16 Number of Kernel Rows (count) 10 Kernel RowType (Distinct, Indistinct) Distinct Kernel-Row Alignment SlightlyCurved (Straight, Slightly Curved, Spiral) Ear Taper (Slight, Average,Extreme) Average Cob Color (standard) Red (14) Cob Color (Munsell) 10 R4/6 KERNEL Type (Flint or Dent) Flint Endosperm Type (description)Normal Starch (3) Yellow Orange Hard Endosperm Color (standard) (8) HardEndosperm Color (Munsell) 7.5 YR 7/8 Kernel Crown Color (standard)Yellow (7) Kernel Crown Color (Munsell) 2.5 Y 8/10

It should be appreciated by one having ordinary skill in the art that,for the quantitative characteristics identified in Table 1, the valuespresented are typical values. These values may vary due to theenvironment and accordingly, other values that are substantiallyequivalent are also within the scope of the invention.

Inbred corn line LLM02BM shows uniformity and stability within thelimits of environmental influence for the traits described in Table 1.Inbred LLM02BM has been self-pollinated and ear-rowed a sufficientnumber of generations with careful attention paid to uniformity of planttype to ensure the homozygosity and phenotypic stability necessary touse in large scale, commercial production. The line has been increasedboth by hand and sib-pollinated in isolated fields with continuedobservations for uniformity. No variant traits have been observed or areexpected in LLM02BM.

The present invention also relates to one or more corn plant parts ofinbred corn plant LLM02BM. Corn plant parts include plant cells, plantprotoplasts, plant cell tissue cultures from which corn plants can beregenerated, intact genomic plant DNA, plant calli, plant clumps, andplant cells that are intact in plants or parts of plants, such asembryos, pollen, ovules, flowers, seeds, kernels, ears, cobs, leaves,husks, stalks, roots, root tips, brace roots, lateral tassel branches,anthers, tassels, glumes, silks, tillers, and the like.

B. Inbred Corn Seed Designated LLM02BM

A corn kernel is composed of four structural parts: (1) the pericarp,which is a protective outer covering (also known as bran or hull); (2)the germ (also known as an embryo); (3) the endosperm; and, (4) the tipcap, which is the point of attachment to the cob. Another aspect of thepresent invention is one or more parts of inbred corn seed LLM02BM, suchas the pericarp of inbred corn seed LLM02BM or the germ and/or theendosperm of inbred corn seed LLM02BM which remain upon removal of thepericarp and adhering remnants of the seed coat.

Inbred corn seed designated LLM02BM may be provided as a substantiallyhomogenous composition of inbred corn seed designated LLM02BM, that is,a composition that consists essentially of inbred corn seed LLM02BM.Such a substantially homogenous composition of inbred corn seed LLM02BMis substantially free from significant numbers of other inbred and/orhybrid seed so that the inbred seed forms from about 90% to about 100%of the total seed. Preferably, a substantially homogenous composition ofthe inbred corn seed contains from about 98.5%, 99%, or 99.5% to about100% of the inbred seed, as measured by seed grow outs. Thesubstantially homogenous composition of inbred corn seed of theinvention may be separately grown to provide substantially homogenouspopulations of inbred corn plants. However, even if a population ofinbred corn plants is present in a field with other different cornplants, such as in a commercial seed-production field of single-crosshybrid corn planted in a ratio of 1 male pollinator row to 4 femaleseed-parent rows, such a population would still be considered to bewithin the scope of the present invention.

Corn yield is affected by the conditions to which seeds and seedlings(young plants grown from seeds) are exposed. Seeds and seedlings may beexposed to one of, or a combination of, for example, cold, drought,salt, heat, pollutants, and disease, all of which are conditions thatpotentially retard or prevent the growth of crops therefrom. Forexample, temperature extremes are typical in the upper Midwest region ofthe United States. Furthermore, diseases evolved from pathogens anddeterioration caused by fungi are potentially harmful to seeds andseedlings. Thus, it is desirable to treat seeds as by coating orimpregnating the seeds with compositions that render the seeds andseedlings grown therefrom more hardy when exposed to such adverseconditions.

Accordingly, another aspect of the present invention relates to a coatedand/or impregnated seed or corn inbred line designated LLM02BM and tocoated and/or impregnated seed derived therefrom. Various agents havebeen used to treat seeds to increase resistance of the plants tostressed conditions, such as cold, drought, salt, and fungi. Such agentsinclude, for example, sodium methylphenyl-pentadienate, trichloroaceticacid, polyoxyalkylene-organo-siloxane block copolymer, 5-aminolevulinicacid, salicylic acid, thiamethoxam, potassium chloride, and polyvinylalcohol and are useful alone, or in combination in the presentinvention.

When pre-treating seeds according to the present invention such asbefore the seeds are planted, the seeds are contacted with thecomposition of interest, as by coating seeds, spraying seeds, andsoaking seeds or a combination thereof, by methods well known to thoseskilled in the art.

C. Deposit Information

Applicants have made a deposit of at least 2,500 seeds of inbred cornplant LLM02BM with the American Type Culture Collection (ATCC),Manassas, Va. 20110 USA, under ATCC Accession No. _(——————). The seedsdeposited with the ATCC on _(——————) were taken from a depositmaintained by Agrigenetics, Inc. d/b/a Mycogen Seeds since prior to thefiling date of this application. Access to this deposit will beavailable during the pendency of the application to the Commissioner ofPatents and Trademarks and persons determined by the Commissioner to beentitled thereto upon request. Upon allowance of any claims in theapplication, the Applicant(s) will maintain and will make this depositavailable to the public pursuant to the Budapest Treaty.

III. Processes of Preparing Novel Corn Plants

A. Novel Inbred Corn Plants Obtained from Inbred LLM02BM

Various breeding schemes may be used to produce new inbred corn linesfrom LLM02BM. In one method, generally referred to as the pedigreemethod, LLM02BM may be crossed with another different corn plant such asa second inbred parent corn plant, which either itself exhibits one ormore selected desirable characteristic(s) or imparts selected desirablecharacteristic(s) to a hybrid combination. Examples of potentiallydesired characteristics include greater yield, better stalks, betterroots, reduced time to crop maturity, better agronomic quality, highernutritional value, higher starch extractability or starchfermentability, resistance and/or tolerance to insecticides, herbicides,pests, heat and drought, and disease, and uniformity in germinationtimes, stand establishment, growth rate, maturity and kernel size. Ifthe two original parents corn plants do not provide all the desiredcharacteristics, then other sources can be included in the breedingpopulation. Elite inbred lines, that is, pure breeding, homozygousinbred lines, can also be used as starting materials for breeding orsource populations from which to develop inbred lines.

Thereafter, resulting seed is harvested and resulting superior progenyplants are selected and selfed or sib-mated in succeeding generations,such as for about 5 to about 7 or more generations, until a generationis produced that no longer segregates for substantially all factors forwhich the inbred parents differ, thereby providing a large number ofdistinct, pure-breeding inbred lines.

In another embodiment for generating new inbred corn plants, generallyreferred to as backcrossing, one or more desired traits may beintroduced into inbred parent corn plant LLM02BM (the recurrent parent)by crossing the LLM02BM plants with another corn plant (referred to asthe donor or non-recurrent parent) which carries the gene(s) encodingthe particular trait(s) of interest to produce F₁ progeny plants. Bothdominant and recessive alleles may be transferred by backcrossing. Thedonor plant may also be an inbred, but in the broadest sense can be amember of any plant variety or population cross-fertile with therecurrent parent. Next, F₁ progeny plants that have the desired traitare selected. Then, the selected progeny plants are crossed with LLM02BMto produce backcross progeny plants. Thereafter, backcross progenyplants comprising the desired trait and the physiological andmorphological characteristics of corn inbred line LLM02BM are selected.This cycle is repeated for about one to about eight cycles, preferablyfor about 3 or more times in succession to produce selected higherbackcross progeny plants that comprise the desired trait and all of thephysiological and morphological characteristics of corn inbred lineLLM02BM listed in Table 1 as determined at the 5% significance levelwhen grown in the same environmental conditions. Exemplary desiredtrait(s) include insect resistance, cytoplasmic male sterility, enhancednutritional quality, waxy starch, herbicide resistance, yield stability,yield enhancement and resistance to bacterial, fungal and viral disease.One of ordinary skill in the art of plant breeding would appreciate thata breeder uses various methods to help determine which plants should beselected from the segregating populations and ultimately which inbredlines will be used to develop hybrids for commercialization. In additionto the knowledge of the germplasm and other skills the breeder uses, apart of the selection process is dependent on experimental designcoupled with the use of statistical analysis. Experimental design andstatistical analysis are used to help determine which plants, whichfamily of plants, and finally which inbred lines and hybrid combinationsare significantly better or different for one or more traits ofinterest. Experimental design methods are used to assess error so thatdifferences between two inbred lines or two hybrid lines can be moreaccurately determined Statistical analysis includes the calculation ofmean values, determination of the statistical significance of thesources of variation, and the calculation of the appropriate variancecomponents. Either a five or a one percent significance level iscustomarily used to determine whether a difference that occurs for agiven trait is real or due to the environment or experimental error. Oneof ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr, Walt, Principles of CultivarDevelopment, p. 261-286 (1987) which is incorporated herein byreference. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions.

This method results in the generation of inbred corn plants withsubstantially all of the desired morphological and physiologicalcharacteristics of the recurrent parent and the particular transferredtrait(s) of interest. Because such inbred corn plants are heterozygousfor loci controlling the transferred trait(s) of interest, the lastbackcross generation would subsequently be selfed to provide purebreeding progeny for the transferred trait(s).

Backcrossing may be accelerated by the use of genetic markers such asSSR, RFLP, SNP or AFLP markers to identify plants with the greatestgenetic complement from the recurrent parent.

Direct selection may be applied where a single locus acts as a dominanttrait, such as the herbicide resistance trait. For this selectionprocess, the progeny of the initial cross are sprayed with the herbicidebefore the backcrossing. The spraying eliminates any plants which do nothave the desired herbicide resistance characteristic, and only thoseplants which have the herbicide resistance gene are used in thesubsequent backcross. In the instance where the characteristic beingtransferred is a recessive allele, it may be necessary to introduce atest of the progeny to determine if the desired characteristic has beensuccessfully transferred. The process of selection, whether direct orindirect, is then repeated for all additional backcross generations.

It should be appreciated by those having ordinary skill in the art thatbackcrossing can be combined with pedigree breeding as where inbredLLM02BM is crossed with another corn plant, the resultant progeny arecrossed back to inbred LLM02BM and thereafter, the resulting progeny ofthis single backcross are subsequently inbred to develop new inbredlines. This combination of backcrossing and pedigree breeding is usefulas when recovery of fewer than all of the LLM02BM characteristics thanwould be obtained by a conventional backcross are desired.

In an additional embodiment of the present invention, new inbred cornplants can be developed by a method generally referred to as haploidbreeding. In this methodology, haploid plants are generated fromdiploid, heterozygous corn plants that result from crossing inbred cornplant LLM02BM with another, different corn plant. Such haploid cornplants may be generated by methods known to those skilled in the artsuch as by culturing haploid anthers or embryos from a diploid plant.Alternately, such haploid corn plant may be generated by crossing thediploid heterozygous corn plant with a corn plant that comprises ahaploid inducing gene, such as the mutant gene “indeterminategametophyte” (ig), which, when present in the female parent results inoffspring with a greatly enhanced frequency of haploids of both maternaland paternal origin. Thereafter, homozygous diploid plants are producedby the doubling of a set of chromosomes (1N) from a haploid plantgenerated by self-pollination such as through use of a doubling agent,such as colchicine, nitrous oxide gas, heat treatment and trifluralin.See, e.g., Wan et al., “Efficient Production of Doubled Haploid PlantsThrough Colchicine Treatment of Anther-Derived Maize Callus”,Theoretical and Applied Genetics, 77:889-892, 1989 and U.S. PatentApplication No. 20030005479 the disclosure of which is expresslyincorporated herein by reference. The technique of haploid breeding isadvantageous because no subsequent inbreeding is required to obtain ahomozygous plant from a heterozygous source. Thus, in another aspect ofthis invention a new inbred corn plant is developed by a method thatincludes the steps of crossing LLM02BM or a hybrid made with LLM02BMwith another inbred corn plant having a propensity to generate haploidsto produce haploid progeny plants, and selecting desirable inbred cornplants from the haploid progeny plants.

The present invention also relates to novel corn plants produced by amethod generally referred to as mutation breeding whereby one or morenew traits may be artificially introduced into inbred line LLM02BM. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation; such as X-rays, Gamma rays (e.g. cobalt60 or cesium 137), neutrons, (product of nuclear fission by uranium 235in an atomic reactor), Beta radiation (emitted from radioisotopes suchas phosphorus 32 or carbon 14), or ultraviolet radiation (preferablyfrom 2500 to 2900 nm), or chemical mutagens (such as base analogues(5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics(streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards,epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones),azide, hydroxylamine, nitrous acid, or acridines. Once a desired traitis observed through mutagenesis and selected, the trait may then beincorporated into existing germplasm by traditional breeding techniques.Details of mutation breeding can be found in “Principals of CultivarDevelopment” Fehr, 1993 Macmillan Publishing Company the disclosure ofwhich is incorporated herein by reference.

The mutagenesis treatment may be applied to various stages of plantdevelopment, including but not limited to cell cultures, embryos,microspores and shoot apices as well as to corn kernels. By way ofexample, pollen may be mixed with a solution of 1 ml EMS and 100 mlsFisher paraffin oil (stock diluted by 1 ml and 15 mls oil solution)every minute for the first 5 minutes and then every five minutes for 45minutes to keep the pollen suspended. Thereafter, the pollen/paraffinoil solution is brushed onto the silks of developing ears. A tassel bagis used to cover the ear to prevent contamination. The ear is picked atmaturity and then resultant seeds or the plants therefrom are screenedfor the desired mutant trait(s).

Once inbred lines are created, the next step is to determine if theinbreds have any value. This is accomplished by techniques of measuringthe combining ability of the new inbred plant, as well as theperformance of the line itself. Combining ability refers to a line'scontribution as a parent when crossed with other lines to form hybrids.Specific combining ability (SCA) refers to the ability of a line tocross to another specific inbred to form a hybrid. General combiningability (GCA) refers to the ability of a line to cross to a wide rangeof lines to form hybrids. The methodology of forming hybrids to evaluatean inbred line's contribution as a parent for the purpose of selectingsuperior lines is interchangeably known as experimental, top or testcrossing.

B. Novel Inbred Plants Obtained from a Hybrid Having Inbred LLM02BM as aParent

In accordance with processes of the present invention, a hybrid planthaving inbred LLM02BM as a parent is crossed with itself or anydifferent corn plant such as an inbred corn plant or a hybrid corn plantto develop a novel inbred line. For example, a hybrid corn plant havinginbred corn plant LLM02BM as a parent may be inbred, i.e., crossed toitself or sib-pollinated, and the resulting progeny each selfed forabout 5 to about 7 or more generations, thereby providing a set ofdistinct, pure-breeding inbred lines wherein each of the lines receivedall of its alleles from the hybrid corn plant having inbred corn plantLLM02BM as a parent. Double haploid methods can also be used to obtainan inbred corn plant that is homozygous at essentially every locus,wherein the inbred corn plant received all of its alleles from thehybrid corn plant having inbred corn plant LLM02BM as a parent. In otherembodiments, a hybrid corn plant having inbred corn plant LLM02BM as aparent is crossed with a different corn plant that may include anyinbred corn plant that is not inbred corn plant LLM02BM, any hybrid cornplant that does not have LLM02BM as a parent, another germplasm source,a haploid or mutation inducing stock, or a trait donor plant, therebyproviding a set of distinct, pure-breeding inbred lines. The resultinginbred lines could then be crossed with other inbred or non-inbred linesand the resulting inbred progeny analyzed for beneficialcharacteristics. In this way, novel inbred lines conferring desirablecharacteristics could be identified.

C. “Chasing Selfs”

Both female and male inbred seed may occasionally be found within acommercial bag of hybrid seed. Chasing the selfs involves identifyinginbred plants within a stand of corn that has been grown from a bag ofhybrid corn seed. Once the seed is planted, the inbred plants may beidentified and selected due to their decreased vigor, i.e., by theirshort stature, narrower leaves, and smaller tassels relative to thehybrid plants that grow from the hybrid seed which predominates in acommercial bag of hybrid seed. By locating the inbred plants, isolatingthem from the rest of the plants, and self-pollinating them (i.e.,“chasing selfs”), a breeder can obtain an inbred line that is identicalto an inbred parent used to produce the hybrid.

Accordingly, another embodiment of the present invention is directed toa method for producing inbred corn plant LLM02BM comprising: (a)planting a collection of seed, such as a collection of seed comprisingseed of a hybrid, one of whose parents is inbred corn plant LLM02BM, thecollection also comprising seed of the inbred; (b) growing plants fromsaid collection of seed; (c) identifying inbred parent plants; (d)controlling pollination in a manner which preserves homozygosity of theinbred parent plant; and, (e) harvesting resultant seed. Step (c) mayfurther comprise identifying plants with decreased vigor, i.e., plantsthat appear less robust than the other plants, or identifying plantsthat have a genetic profile in accordance with the genetic profile ofLLM02BM, such as an SSR genetic profile in accordance with Table 5herein. Corn plants capable of expressing substantially all of thephysiological and morphological characteristics of inbred corn plantLLM02BM include corn plants obtained by chasing selfs from a bag ofhybrid seed.

One having skill in the art will recognize that once a breeder hasobtained inbred corn plant LLM02BM by chasing selfs from a bag of hybridseed, the breeder can then produce new inbred plants such as bysib-pollinating, i.e., crossing the inbred corn plant LLM02BM withanother inbred corn plant LLM02BM, or by crossing the inbred corn plantLLM02BM with a hybrid corn plant obtained by growing the collection ofseed.

IV. Novel Hybrid Plants

A. Novel Hybrid Seeds and Plants

In yet another aspect of the invention, processes are provided forproducing corn seeds or plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plantwherein at least one of the first parent corn plant or the second parentcorn plant is inbred parent corn plant LLM02BM. In some embodiments ofthe present invention, the first inbred corn plant is LLM02BM and is afemale and in other embodiments the first inbred corn plant is LLM02BMand is a male. These processes may be further exemplified as processesfor preparing hybrid corn seed or plants, wherein a first inbred cornplant is crossed with a second corn plant of a different, distinctvariety to provide a hybrid that has, as one of its parents, the inbredcorn plant variety LLM02BM. In this case, a second inbred variety isselected which confers desirable characteristics when in hybridcombination with the first inbred line. In these processes, crossingwill result in the production of seed. The seed production occursregardless whether the seed is collected.

Any time the inbred corn plant LLM02BM is crossed with another,different corn inbred, a first generation (F₁) corn hybrid plant isproduced. As such, an F₁ hybrid corn plant may be produced by crossingLLM02BM with any second inbred corn plant. Therefore, any F₁ hybrid cornplant or corn seed which is produced with LLM02BM as a parent is part ofthe present invention.

When inbred corn plant LLM02BM is crossed with another inbred plant toyield a hybrid, the original inbred can serve as either the maternal orpaternal plant with basically, the same characteristics in the hybrids.Occasionally, maternally inherited characteristics may expressdifferently depending on the decision of which parent to use as thefemale. However, often one of the parental plants is preferred as thematernal plant because of increased seed yield and preferred productioncharacteristics, such as optimal seed size and quality or ease of tasselremoval. Some plants produce tighter ear husks leading to more loss, forexample due to rot, or the ear husk may be so tight that the silk cannotcompletely push out of the tip preventing complete pollination resultingin lower seed yields. There can be delays in silk formation whichdeleteriously affect timing of the reproductive cycle for a pair ofparental inbreds. Seed coat characteristics can be preferable in oneplant which may affect shelf life of the hybrid seed product. Pollen canshed better by one plant, thus rendering that plant as the preferredmale parent. It is generally preferable to use LLM02BM as the maleparent.

In embodiments of the present invention, the first step of “crossing”the first and the second parent corn plants comprises planting,preferably in pollinating proximity, seeds of a first inbred corn plantand a second, distinct inbred corn plant. As discussed herein, the seedsof the first inbred corn plant and/or the second inbred corn plant canbe treated with compositions that render the seeds and seedlings growntherefrom more hardy when exposed to adverse conditions.

A further step comprises cultivating or growing the seeds of the firstand second parent corn plants into plants that bear flowers. If theparental plants differ in timing of sexual maturity, techniques may beemployed to obtain an appropriate nick, i.e., to ensure the availabilityof pollen from the parent corn plant designated the male during the timeat which silks on the parent corn plant designated the female arereceptive to the pollen. Methods that may be employed to obtain thedesired nick include delaying the flowering of the faster maturingplant, such as, but not limited to delaying the planting of the fastermaturing seed, cutting or burning the top leaves of the faster maturingplant (without killing the plant) or speeding up the flowering of theslower maturing plant, such as by covering the slower maturing plantwith film designed to speed germination and growth or by cutting the tipof a young ear shoot to expose silk.

In a preferred embodiment, the corn plants are treated with one or moreagricultural chemicals as considered appropriate by the grower.

A subsequent step comprises preventing self-pollination orsib-pollination of the plants, i.e., preventing the silks of a plantfrom being fertilized by any plant of the same variety, including thesame plant. This is preferably done in large scale production bycontrolling the male fertility, e.g., treating the flowers so as toprevent pollen production or alternatively, using as the female parent amale sterile plant of the first or second parent corn plant (i.e.,treating or manipulating the flowers so as to prevent pollen production,to produce an emasculated parent corn plant or using as a female, acytoplasmic male sterile version of the corn plant). This control mayalso be accomplished in large scale production by physical removal ofthe tassel from the female plant, either by pulling the tassel by hand,cutting with a rotary cutter, or pulling with a mechanical tasselpulling machine. In small scale production, corn breeder's shoot bags,usually plastic or glassine, applied to cover the ear shoot prior to theextrusion of silks provide effective control of unwantedself-pollination or sib-pollination.

Yet another step comprises allowing cross-pollination to occur betweenthe first and second parent corn plants. When the plants are not inpollinating proximity, this is done by placing a bag, usually paper,over the tassels of the first plant and another shoot bag over the earshoot, prior to the extrusion of silk, of the incipient ear on thesecond plant. The bags are left in place usually overnight. Since pollenstops shedding each day and loses viability and new pollen is shed eachmorning, this assures that the silks are not pollinated from otherpollen sources, that any stray pollen on the tassels of the first plantis dead, and that the only pollen transferred comes from the firstplant. The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels and removedfrom the first plant. Finally, in one continuous motion, the shoot bagis removed from the silks of the incipient ear on the second plant, andthe pollen bag containing the captured pollen is placed over the silksof the incipient ear of the second plant, shaken again to disperse thecaptured pollen, and left in place covering the developing ear toprevent contamination from any unwanted fresh airborne pollen. In largescale production, crossing is accomplished by isolated open-pollinatedcrossing fields whereby corn plants of the parent designated as thefemale, which are controlled for male fertility, are allowed to bepollinated by other plants of a different corn type where such plantsare adjacent to the plants designated as the female parent.

A further step comprises harvesting the seeds, near or at maturity, fromthe ear of the plant that received the pollen. In a particularembodiment, seed is harvested from the female parent plant, and whendesired, the harvested seed can be grown to produce a first generation(F₁) hybrid corn plant.

Yet another step comprises drying and conditioning the seeds, includingthe treating, sizing (or grading) of seeds, and packaging for sale togrowers for the production of grain or forage. As with inbred seed, itmay be desirable to treat hybrid seeds with compositions that render theseeds and seedlings grown therefrom more hardy when exposed to adverseconditions. Mention should be made that resulting hybrid seed is sold togrowers for the production of grain and forage and not for breeding orseed production.

Still further, the present invention provides a hybrid corn plantproduced by growing the harvested seeds produced on the male-sterileplant as well as grain produced by the hybrid corn plant.

A single cross hybrid is produced when two different inbred parent cornplants are crossed to produce first generation F₁ hybrid progeny.Generally, each inbred parent corn plant has a genotype whichcomplements the genotype of the other inbred parent. Typically, the F₁progeny are more vigorous then the respective inbred parent corn plants.This hybrid vigor, or heterosis, is manifested in many polygenic traits,including markedly improved yields and improved stalks, roots,uniformity and insect and disease resistance. It is for this reason thatsingle cross F₁ hybrids are generally the most sought after hybrid. Athree-way, or modified single-cross hybrid is produced from three inbredlines (or synthetics) where two of the inbred lines are crossed (A×B)and then the resulting F₁ hybrid is crossed with the third inbred(A×B)×C, as where a modified female is used in the cross. A modifiedfemale provides an advantage of improved seed parent yield whereas amodified male improves pollen flow. A double cross hybrid is producedfrom four inbred lines crossed in pairs (A×B and C×D), thereby resultingin two F₁ hybrids that are crossed again. Double cross hybrids are morecommon in countries wherein less demand exists for higher yieldingsingle cross hybrids. Synthetic populations or crosses are developed bycrossing two or more inbred lines (or hybrids, or germplasm sources)together and then employing one of many possible techniques to randommate the progeny. Random mating the progeny is any process used by plantbreeders to make a series of crosses that will create a new germplasmpool from which new breeding lines can be derived. Much of the hybridvigor exhibited by F₁ hybrids is lost in the next generation (F₂).Consequently, seed from hybrids are not typically used for plantingstock.

The utility of the inbred plant LLM02BM also extends to crosses withspecies other than the mays species, such as diploperennis, luxurians,and perennis. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schleachne, Polytoca, Chionachne, and Trilobachne. Of these, Zea andTripsacum, are most preferred. Varieties of the grain sorghum Sorghumbicolor (L.) Moench can be crossed with inbred corn line LLM02BM.

B. Physical Description of F₁ Hybrids and F₁ Hybrid Comparison

As mentioned above, testcross hybrids are progressively eliminatedfollowing detailed evaluations of their phenotype, including formalcomparisons with other commercially successful hybrids. Researchsmall-plot trials and commercial strip trials are used to compare thephenotypes of hybrids grown in as many environments as possible. Theyare performed in many environments to assess overall performance of thenew hybrids and to select optimum growing conditions. Because the cornis grown in close proximity, differential effects of environmentalfactors that affect gene expression, such as moisture, temperature,sunlight, and pests, are minimized. For a decision to be made to advancea hybrid, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create value for some applications or markets. Sometestcross hybrids are eliminated despite being similarly competitiverelative to the current commercial hybrids because of the cost to bringa new hybrid to market requires a new product to be a significantimprovement over the existing product offering. Such hybrids may also belicensed to other parties who have a need in their commercial productportfolio.

The present invention provides F₁ hybrid corn plants obtained from thecorn plant LLM02BM. The performance characteristics of an exemplaryhybrid produced using LLM02BM as one inbred parent are set forth inTables 2 and 3. The results in Table 2 compare hybrid performance ofinbred LLM02BM and another elite inbred, when each of these inbreds iscrossed to the same tester line. The results in Table 3 shows hybridperformance for the hybrid made with LLM02BM and the tester linecompared with selected commercial hybrids including Mycogen F2F714 andMycogen F2F797.

TABLE 2 Comparison of Corn Inbred LLM02BM to Another Inbred in Hybridswith Common Tester Line LLM02BM × Inbred C × Characteristic Tester lineTester line Biomass (tons/ac) 25.72 25.78 Silage Moisture (%) 65.3867.67 NDFD (%) 68.88 69.79 Starch (%) 26.35 23.64 TDMD (%) 83.60 82.78Crude Protein (%) 7.02 6.70 Plant Height (cm) 269.20 293.10 Ear Height(cm) 134.60 148.30 Stalk Lodging (%) 0.47 0.00 Root Lodging (%) 0.002.39 Population (plants/ac) 30,650 29,560 Environments (count) 22 20Plots/mean (count) 22 20

TABLE 3 Hybrid From LLM02BM Compared With Other CommercialHybridsLLM02BM × Mycogen Mycogen Characteristic Tester line A F2F714 F2F797Biomass (tons/ac) 25.72 24.00 23.00 Silage Moisture (%) 65.38 63.0065.00 NDFD (%) 68.88 65.90 67.90 Starch (%) 26.35 26.00 25.00 PlantHeight (cm) 269.20 287.00 330.00 Ear Height (cm) 134.60 155.00 168.00Stalk Lodging (%) 0.47 1.50 14.60 Root Lodging (%) 0.00 1.20 18.00Population 30,650 29,880 25,850 (plants/ac) Environments 22 30 14(count) Plots/mean (count) 22 30 14

V. Novel LLM02BM-Derived Plants

All plants produced using inbred corn plant LLM02BM as a parent arewithin the scope of this invention, including plants derived from inbredcorn plant LLM02BM. This includes plants essentially derived from inbredLLM02BM with the term “essentially derived variety” having the meaningascribed to such term in 7 U.S.C. §2104(a)(3) of the Plant VarietyProtection Act, which definition is hereby incorporated by reference.This also includes progeny plant and parts thereof with at least oneancestor that is inbred corn plant LLM02BM and more specifically wherethe pedigree of this progeny includes 1, 2, 3, 4, and/or 5 or crosspollinations to inbred corn plant LLM02BM, or a plant that has LLM02BMas a progenitor. All breeders of ordinary skill in the art maintainpedigree records of their breeding programs. These pedigree recordscontain a detailed description of the breeding process, including alisting of all parental lines used in the breeding process andinformation on how such line was used. Thus, a breeder would know ifLLM02BM were used in the development of a progeny line, and would alsoknow how many breeding crosses to a line other than LLM02BM were made inthe development of any progeny line. A progeny line so developed maythen be used in crosses with other, different, corn inbreds to producefirst generation F₁ corn hybrid seeds and plants with superiorcharacteristics.

Accordingly, another aspect of the present invention is methods forproducing an inbred corn line LLM02BM-derived corn plant. This methodfor producing a LLM02BM-derived corn plant, comprises: (a) crossinginbred corn plant LLM02BM with a second corn plant to yield progeny cornseed; and, (b) growing the progeny corn seed, (under plant growthconditions), to yield the LLM02BM-derived corn plant. Such methods mayfurther comprise the steps of: (c) crossing the LLM02BM-derived cornplant with itself or another corn plant to yield additionalLLM02BM-derived progeny corn seed; (b) growing the progeny corn seed ofstep (d) (under plant growing conditions), to yield additionalLLM02BM-derived corn plants; and (e) repeating the crossing and growingsteps of (c) and (d) from 0 to 7 times to generate furtherLLM02BM-derived corn plants. Still further, this may comprise utilizingmethods of haploid breeding and plant tissue culture methods to deriveprogeny of the LLM02BM-derived corn plant.

VI. Tissue Cultures and In Vitro Regeneration of Corn Plants

As is well known in this art, tissue culture of corn may be used for thein vitro regeneration of a corn plant. Accordingly, a further aspect ofthe invention relates to tissue cultures of the inbred corn plantdesignated LLM02BM, to tissue cultures of hybrid and derived corn plantsobtained from LLM02BM, to plants obtained from such tissue cultures andto the use of tissue culture methodology in plant breeding. The term“tissue culture” includes a composition comprising isolated cells of thesame type, isolated cells of a different type, or a collection of suchcells organized into parts of a plant. Exemplary tissue cultures areprotoplasts, calli and plant cells that are intact in plants or parts ofplants, such as embryos, pollen, flowers, kernels, ears, cobs, leaves,husks, stalks, roots, root tips, anthers, silk, and the like. In apreferred embodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts.

A. Immature Embryo Culture

To obtain immature embryos for callus culture initiation, ears areharvested from a corn plant, e.g., an inbred corn plant LLM02BM, ahybrid corn plant having LLM02BM as a parent or a LLM02BM-derived cornplant, approximately 9-10 days post-pollination. Initially, harvestedears are surface sterilized, as by scrubbing with Liqui-Nox® soap,immersion in 70% ethanol for 2-3 minutes, followed by immersion in 20%commercial bleach (0.1% sodium hypochlorite) for about 30 minutes.

Ears are rinsed in sterile, distilled water, and immature zygoticembryos are aseptically excised with the aid of a dissection microscopeand cultured on a suitable initiation medium. One having ordinary skillin the art would understand that explants from other tissues, such asimmature tassel tissue, intercalary meristems and leaf bases, apicalmeristems, and immature ears may also be the subject of callus cultureinitiation. Tissue culture media typically, contain amino acids, salts,sugars, hormones, and vitamins. Most of the media employed to regenerateinbred and hybrid plants have some similar components; the media differin the composition and proportions of their ingredients depending on theparticular application envisioned. Examples of media suitable forculture of plants cells include, but are not limited to an N6 medium, anMS media, or modifications thereof. A preferred media is a 15Ag10 medium(N6 Complete Medium, PhytoTechnology Laboratories C167), 1.0 mg/L 2,4-D,20 g/L sucrose, 100 mg/L casein hydrolysate (enzymatic digest), 25 mML-proline, 10 mg/L AgNO₃, 2.5 g/L Gelrite, pH 5.8, for about 2-3 weekswith the scutellum facing away from the medium. Tissue showing theproper morphology, Welter, et al., Plant Cell Rep. 14:725-729 (1995), isselectively transferred at biweekly intervals onto fresh 15Ag10 mediumfor about 6 weeks and then is transferred to “4” medium (N6 CompleteMedium Phyto 167), 1.0 mg/L 2,4-D, 20 g/L sucrose, 100 mg/L caseinhydrolysate (enzymatic digest), 6 mM L-proline, 2.5 g/L Gelrite, pH 5.8,at bi-weekly intervals for approximately 2 months. Hormones other thanan auxin such as 2,4-D may be employed including dicamba, NAA, BAP,2-NCA, ABA, and picloram. Modifications of these and other basic mediamay facilitate growth of recipient cells at specific developmentalstages.

Regeneration is initiated by transferring callus tissue to acytokinin-based induction medium, “28” (MS Salts, 30 g/L sucrose, 5 mg/LBAP, 0.025 mg/L 2,4-D at pH of 5.7.) Cells are allowed to grow in lowlight (13 μEM⁻²s⁻¹) for one week then higher light (40 μEM⁻²s⁻¹) forabout another week. Regeneration of plants is completed by the transferof mature and germinating embryos to a hormone-free medium, followed bythe transfer of developed plantlets to soil and growth to maturity. In apreferred embodiment, the cells are transferred to a hormone-freeregeneration medium, “28” lacking plant growth regulators. Thereafter,small (3-5 cm) plantlets are removed and placed into 150×25 mm culturetubes containing SHGA (Schenk and Hilldebrandt salts: 10 g/L sucrose, 1g/L myo-inositol, 2 g/L Gelrite, pH 5.8) medium.

Once plantlets develop a sufficient root and shoot system, they aretransplanted to 4-inch pots containing approximately 0.1 kg of METRO-MIX360 soil in a growth room or greenhouse. They are grown with a 16-hphotoperiod supplemented by a combination of high pressure sodium andmetal halide lamps, and are watered as needed with a combination ofthree different Miracle Grow fertilizer formulations. At the 3-5 leafstage, plants are transferred to five gallon pots containingapproximately 4 kg METRO-MIX 360.

Primary regenerants (R₁ plants) are self- or sib-pollinated after anadditional 6-10 weeks in five gallon pots, and R₁ seed is collected at40-45 days post-pollination. Alternately, when self- or sib-pollinationsare not possible, plants may be outcrossed to elite inbreds.

It will be appreciated by those of ordinary skill in the art thatregenerable cultures, including Type I and Type II cultures, may beinitiated from immature embryos using other methods, such as thosedescribed in, for example, PCT Application WO 95/06128, the disclosureof which is incorporated herein by reference in its entirety.

B. Additional Tissue Cultures and Regeneration

Other means for preparing and maintaining plant tissue cultures are wellknown in the art. By way of example, a tissue culture comprising organssuch as tassels or anthers and pollen (microspores) have been used toproduce regenerated plants (U.S. Pat. Nos. 5,322,789 and 5,445,961).Also, meristematic cells (i.e., plant cells capable of continual celldivision and characterized by an undifferentiated cytologicalappearance, normally found at growing points or tissues in plants suchas root tips, stem apices, lateral buds, etc.) can be cultured toregenerate fertile plants (U.S. Pat. No. 5,736,369 the disclosure ofwhich is specifically incorporated herein by reference).

VII. Male Sterility

Methods for controlling male fertility in corn plants offer theopportunity for improved plant breeding, particularly for thedevelopment of corn hybrids which require the implementation of a malesterility system to prevent the inbred parent plants fromself-pollination.

Accordingly, another aspect of the present invention is male-sterileinbred corn plants designated LLM02BM and the production of hybrid cornseed using a male sterility system with such inbred female parent plantsthat are male sterile. In the event that inbred corn line LLM02BM isemployed as the female parent, LLM02BM can be rendered male-sterile by,for example, removing the tassels or “detasseling” LLM02BM parentalplants either manually or by machine. By way of example, alternatestrips of two corn inbreds may be planted in a field followed by manualor mechanical removal of the pollen-bearing tassels from the designatedfemale inbred. Provided that the female inbreds are sufficientlyisolated from foreign corn pollen sources, the ears of the detasseledinbred will be fertilized only from the other male inbred, and theresulting seed will therefore be hybrid seed.

The laborious and occasionally unreliable detasseling process can beminimized by using cytoplasmic male-sterile (CMS) inbreds. Plants of aCMS inbred are male sterile as a result of factors resulting fromcytoplasmic as opposed to the nuclear genome. Thus, this characteristicis inherited exclusively through the female parent in corn plants sinceCMS plants are fertilized with pollen from another inbred that is notmale-sterile. Pollen from the second inbred may or may not contributegenes that make the hybrid plants male-fertile. Seed from detasseledfertile corn and CMS produced seed of the same hybrid can be blended toinsure that adequate pollen loads are available for fertilization whenthe hybrid plants are grown. Conventional backcrossing methodology maybe used to introgress the CMS trait into inbred LLM02BM.

Alternatively, haploid breeding methods may also be employed to convertinbred LLM02BM to CMS sterility. Haploids are plants which contain onlyone-half of the chromosome number present in diploid somatic cells,which are cells other than haploid cells such as those found in thegerm. There are a few stocks or genetic systems in corn which are knownto generate haploids spontaneously. For example, plants are known whichpossess an indeterminate gametophyte (ig) gene (Kermicle 1969 Science166:1422-1424) which generate haploids. Additionally, a line known asStock 6 (See, Birchler, J. A., “Practical Aspects of HaploidProduction,” The Corn Handbook, Freeling and Walbot (eds). pp. 386-388(1996)) possesses a propensity to generate haploids. Moreover, RWS(Roeber and Geiger 2001, submitted to Crop Science), KEMS (Deimling,Roeber, and Geiger, 1997, Vortr. Pflanzenzuchtg 38:203-224), or KMS andZMS (Chalyk, Bylich & Chebotar, 1994, MNL 68:47; Chalyk & Chebotar,2000, Plant Breeding 119:363-364) also represent inducer lines which beused to produce haploid plants from any genotype. In a preferred aspectof the present invention, cytoplasmic male sterile female plants whichpossess the ig gene may be used to facilitate the generation ofcytoplasmic male sterile inbred versions of LLM02BM of the presentinvention because the selection of haploids and subsequent doublingpermits corn breeders to reach homogeneity more quickly and completely.

Detasseling can also be avoided by the use of chemically induced malesterility in the production of hybrid corn seed. Chemicals that inducemale sterility include gametocides, pollen suppressants, and chemicalhybridizing agents. The general procedure is to use a foliar spraybefore flowering, which inhibits production of viable pollen, but doesnot injure the pistillate reproductive organs or affect seeddevelopment. If the treatment is successful and all of the pollenkilled, self-pollination will not occur in the treated plants, but theflowers will set seed freely from cross-pollination. In such a case, theparent plants used as the male may either not be treated with thechemical agent or may include a genetic factor which causes resistanceto the sterilizing effects of the chemical agent. The use of chemicallyinduced male sterility affects fertility in the plants only for thegrowing season in which the gametocide is applied (see Carlson, GlennR., U.S. Pat. No. 4,963,904).

A further method for controlling male sterility includes the use ofgenes conferring male sterility, such as those disclosed in U.S. Pat.Nos. 3,861,709, 3,710,511, 4,654,465, 5,625, 132, and 4,727,219, each ofthe disclosures of which are specifically incorporated herein byreference in their entirety. Both inducible and non-inducible malesterility genes can increase the efficiency with which hybrids are made,in that they eliminate the need to physically emasculate the corn plantused as a female in a given cross.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 and chromosomal translocations as described byPatterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. These and allpatents referred to are incorporated by reference. In addition to thesemethods, U.S. Pat. No. 5,432,068, discloses a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility; silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on”, the promoter, which in turnallows the gene which confers male fertility to be transcribed.

Other methods of conferring genetic male sterility exist in the art.These methods use a variety of approaches such as delivering into theplant a gene encoding a cytotoxic substance associated with a maletissue specific promoter or an antisense system in which a gene criticalto fertility is identified and an antisense to that gene is inserted inthe plant (see Fabinjanski, et. al, EPO89/3010153.8 publication no 329,308 and PCT application PCT/CA90/00037 published as WO 90/08828).

The presence of a male-fertility restorer gene results in the productionof a fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the corn plant is used, e.g., forsilage, but in most cases, the seeds will be deemed the most valuableportion of the crop, so fertility of the hybrids in these crops must berestored. Therefore, one aspect of the present invention concerns inbredcorn plant LLM02BM comprising a single gene capable of restoring malefertility in an otherwise male-sterile inbred or hybrid plant. Examplesof male-sterility genes and corresponding restorers which could beemployed within the inbred of the invention are well known to those ofskill in the art of plant breeding and are disclosed in, for example,U.S. Pat. Nos. 5,530,191, 5,689,041, 5,741,684, and 5,684,242, thedisclosures of which are each specifically incorporated herein byreference in their entirety.

VIII. Corn Transformation

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and to expressforeign genes, or additional, or modified versions of native orendogenous genes (perhaps driven by different promoters) to alter thetraits of a plant in a specific manner. Such foreign, additional and/ormodified genes are referred to herein collectively as “transgenes.” Thepresent invention, in particular embodiments, also relates totransformed versions of the claimed inbred corn line LLM02BM containingone or more transgenes.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement. The expression vector may contain one or more such operablylinked gene/regulatory element combinations. The vector(s) may be in theform of a plasmid, and can be used, alone or in combination with otherplasmids, to provide transformed corn plants, using transformationmethods as described below to incorporate transgenes into the geneticmaterial of the corn plant(s).

A. Expression Vectors for Corn Transformation/Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element that allows transformed cells containing themarker to be either recovered by negative selection, i.e., inhibitinggrowth of cells that do not contain the selectable marker gene, or bypositive selection, i.e., screening for the product encoded by thegenetic marker. Many commonly used selectable marker genes for planttransformation are well known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or a herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from a bacterialsource, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A. 80: 4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol. 5: 299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet. 210: 86 (1987), Svab et al.,Plant Mol. Biol. 14: 197 (1990), Hille et al., Plant Mol. Biol. 7: 171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or bromoxynil. Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) andStalker et al., Science 242: 419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67(1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant CellRep. 8: 643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), 3-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5: 387 (1987), Teeri et al., EMBO J. 8: 343(1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84: 131 (1987), DeBlock et al., EMBO J. 3: 1681 (1984). Another approach to theidentification of a relatively rare transformation events has been useof a gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247: 449(1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes Publication 2908, Imagene Green™, p. 1-4 (1983) and Naleway etal., J. Cell Biol. 115: 151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263: 802 (1994). GFP and mutants of GFPmay be used as screenable markers.

B. Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissues arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control or is induced inresponse to chemical or hormonal stimuli. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Examples of chemicalsthat induce expression including salicyclic acid and ABA.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions and in all cells.

1. Inducible Promoters

An inducible promoter is operably linked to a gene for expression incorn. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. With an inducible promoter the rate oftranscription increases in response to an inducing agent. Any induciblepromoter can be used in the instant invention. A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone.

2. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression incorn or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in corn. Many different constitutive promoters can beused in the present invention. Exemplary constitutive promoters include,but are not limited to, the promoters from plant viruses such as the 35Spromoter from CaMV and the promoters from such genes as rice actin,maize ubiquitin, and corn H3 histone. Also, the ALS promoter, aXbaI/NcoI fragment 5′ to the Brassica napus ALS3 structural gene (or anucleotide sequence that has substantial sequence similarity to theXbaI/NcoI fragment) represents a particularly useful constitutivepromoter.

3. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin corn. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in corn. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue. Any tissue-specific or tissue-preferred promoter can beutilized in the instant invention. Exemplary tissue-specific ortissue-preferred promoters include, but are not limited to, aseed-preferred promoter such as that from the phaseolin gene; aleaf-specific and light-induced promoter such as that from cab orrubisco; an anther-specific promoter such as that from LAT52; a pollenspecific promoter such as that from Zm13 or a microspore-preferredpromoter such as that from apg.

C. Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Any signal sequence known in the art is contemplated by the presentinvention.

D. Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods.

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is corn. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, and Simple Sequence Repeats (SSR) which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Glick and Thompson, METHODSIN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 269-284 (CRC Press, BocaRaton, 1993). Map information concerning chromosomal location is usefulfor proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

(a) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

(b) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection (Rockville,Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

(c) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

(d) A vitamin-binding protein such as avidin. See PCT applicationUS93/06487 the contents of which are hereby incorporated by. Theapplication teaches the use of avidin and avidin homologues aslarvicides against insect pests.

(e) An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793(1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huubet al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci.Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).

(f) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(g) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm 163: 1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

(h) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116: 165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

(i) An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(j) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule, forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

(k) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a corn calmodulin cDNA clone.

(l) A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

(m) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(n) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(o) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(p) A virus-specific antibody. See, for example, Tavladoraki et al,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonate. See Lamb et al., Bio/Technology10: 1436 (1992). The cloning and characterization of a gene whichencodes a bean endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2: 367 (1992).

(r) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to a Herbicide, for Example:

(a) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Mild et al., Theor. Appl. Genet. 80: 449(1990), respectively.

(b) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phospinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Accl-S1, Accl-S2 and Accl-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(c) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

(a) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89: 2624 (1992).

(b) Decreased phytate content:

(i) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(ii) A gene could be introduced that reduces phytate content. In corn,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor corn mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35: 383 (1990).

(iii) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtillus levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α-amylase), Elliot et al., Plant Molec.Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes),Sogaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102: 1045 (1993) (corn endosperm starch branching enzyme II).

E. Methods for Corn Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, inc., Boca Raton,1993) pages 89-119.

1. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant. Sci. 10: 1 (1991). Descriptions of Agrobacterium vector systemsand methods for Agrobacterium-mediated gene transfer are provided byGruber et al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8: 238 (1989). See also, U.S. Pat. No. 5,591,616, issued Jan. 7,1997.

2. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and corn. Hiei etal., The Plant Journal 6: 271-282 (1994); U.S. Pat. No. 5,591,616,issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm (See e.g., U.S. Pat. No.5,550,318; U.S. Pat. No. 5,736,369, U.S. Pat. No. 5,538,880; and PCTPublication WO 95/06128). The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al, Part. Sci. Technol. 5: 27 (1987),Sanford, J. C., Trends Biotech. 6: 299 (1988), Klein et al.,Bio/Technology 6: 559-563 (1988), Sanford, J. C., Physiol Plant 79: 206(1990), Klein et al., Biotechnology 10: 268 (1992). In corn, severaltarget tissues can be bombarded with DNA-coated microprojectiles inorder to produce transgenic plants, including, for example, callus (TypeI or Type II), immature embryos, and meristematic tissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199: 161 (1985) and Draper et al., Plant Cell Physiol. 23: 451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. U.S. Pat. No. 5,384,253 and Donn et al. In Abstracts ofVIIth International Congress on Plant Cell and Tissue Culture IAPTC,A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4: 1495-1505 (1992) andSpencer et al., Plant Mol. Biol. 24: 51-61 (1994).

Other methods which have been described for the genetic transformationof corn include, electrotransformation (U.S. Pat. No. 5,371,003) andsilicon carbide fiber-mediated transformation (U.S. Pat. No. 5,302,532and U.S. Pat. No. 5,464,765).

Following transformation of corn target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art. Forexample, transformed corn immature embryos.

The foregoing methods for transformation would typically be used forproducing transgenic inbred lines. Transgenic inbred lines could then becrossed, with another (non-transformed or transformed) inbred line, inorder to produce a transgenic hybrid corn plant. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-elite lineinto an elite line, or from a hybrid corn plant containing a foreigngene in its genome into a line or lines which do not contain that gene.

IX. Genetic Complements

In addition to phenotypic observations, a plant can also be described byits genotype. The genotype of a plant can be described through a geneticmarker profile which can identify plants of the same variety, a relatedvariety or be used to determine or to validate a pedigree. Geneticmarker profiles can be obtained by techniques such as RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), SimpleSequence Repeats (SSRs) which are also referred to as Microsatellites,and Single Nucleotide Polymorphisms (SNPs), Isozyme Electrophoresis andIsolelectric Focusing. For example, see Berry, Don, et al., “AssessingProbability of Ancestry Using Simple Sequence Repeat Profiles:Applications to Corn Hybrids and Inbreds”, Genetics, 2002, 161:813-824,which is incorporated by reference herein in its entirety.

Particular markers used for these purposes are not limited to the set ofmarkers disclosed herewithin, but are envisioned to include any type ofgenetically stable marker and marker profile which provides a means ofdistinguishing varieties. In addition to being used for identificationof inbred parents, a hybrid produced through the use of LLM02BM or itsparents, and identification or verification of the pedigree of progenyplants produced through the use of LLM02BM, the genetic marker profileis also useful in breeding and developing backcross conversions.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. The phrase“simple sequence repeats” or “SSR” refers to di-, tri- ortetra-nucleotide repeats within a genome. The repeat region may vary inlength between genotypes while the DNA flanking the repeat is conservedsuch that the primers will work in a plurality of genotypes. Apolymorphism between two genotypes represents repeats of differentlengths between the two flanking conserved DNA sequences. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR). The PCR® detection is done by theuse of two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA followed by DNA amplification. This step involvesrepeated cycles of heat denaturation of the DNA followed by annealing ofthe primers to their complementary sequences at low temperatures, andextension of the annealed primers with DNA polymerase. Size separationof DNA fragments on agarose or polyacrylamide gels followingamplification, comprises the major part of the methodology.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All such publications, patents and patentapplications are incorporated by reference herein to the same extent asif each was specifically and individually indicated to be incorporatedby reference herein.

The foregoing invention has been described in some detail by way ofillustration and example for purposes of clarity and understanding.However, it should be appreciated by those having ordinary skill in theart that certain changes and modifications such as single genemodifications and mutations, somoclonal variants, variant individualsselected from large populations of the plants of the instant inbred andthe like may be practiced within the scope of the invention, as limitedonly by the scope of the appended claims, without departing from thetrue concept, spirit, and scope of the invention.

What is claimed is:
 1. Seed of corn inbred line designated LLM02BM, or apart thereof, representative seed of the line having been depositedunder ATCC Accession No. _(——————).
 2. The seed or part thereof of claim1, wherein the part is selected from the group consisting of pericarp,germ and endosperm.
 3. The seed or part thereof of claim 1, wherein theseed further comprises a coating.
 4. A substantially homogenouscomposition of the corn seed of claim
 1. 5. A method for producing cornseed, said method comprising the steps of: (a) planting seed of claim 1in proximity to itself or to different seed from a same family or line;(b) growing plants from the seed under pollinating conditions; and, (c)harvesting resultant seed.
 6. Corn seed produced by the method of claim5.
 7. The method of claim 5, further comprising pre-treating the seed ofclaim 1 before performing step (a).
 8. The method of claim 5, furthercomprising treating the growing plants or soil surrounding the growingplants with an agricultural chemical.
 9. A corn plant produced bygrowing the seed of claim
 1. 10. A part of the corn plant of claim 9,selected from the group consisting of an intact plant cell, a plantprotoplast, an embryo, a pollen, an ovule, a flower, a kernel, a seed,an ear, a cob, a leaf, a husk, a stalk, a root, a root tip, a braceroot, a lateral tassel branch, an anther, a tassel, a glume, a tillerand a silk.
 11. Pollen of the plant of claim
 9. 12. An ovule of theplant of claim
 9. 13. A corn plant, or a part thereof, having all thephysiological and morphological characteristics of the corn plant ofclaim
 9. 14. A substantially homogenous population of corn plants ofclaim
 9. 15. The substantially homogenous population of corn plants ofclaim 14, wherein the population is present in a field and the fieldfurther comprises other, different corn plants.
 16. A method forproducing a corn plant, comprising: (a) crossing inbred corn plantLLM02BM, representative seed of the line having been deposited underATCC Accession No. _(——————), with another different corn plant to yieldprogeny corn seed.
 17. The method of claim 16, wherein the other,different corn plant is an inbred corn plant.
 18. The method of claim16, further comprising: (b) growing the progeny corn seed from step (a)under self-pollinating or sib-pollinating conditions for about 5 toabout 7 generations; and (c) harvesting resultant seed.
 19. The methodof claim 16, further comprising selecting plants obtained from growingat least one generation of the progeny corn seed for a desirable trait.20. A method of introducing a desired trait into corn inbred lineLLM02BM comprising: (a) crossing LLM02BM plants with plants of anothercorn line that comprise a desired trait to produce F₁ progeny plants;(b) selecting F₁ progeny plants that have the desired trait; (c)crossing selected progeny plants with LLM02BM plants to producebackcross progeny plants; (d) selecting for backcross progeny plantsthat comprise the desired trait and physiological and morphologicalcharacteristics of corn inbred line LLM02BM; and (e) performing steps(c) and (d) one or more times in succession to produce the selected orhigher backcross progeny plants that comprise the desired trait and allof the physiological and morphological characteristics of corn inbredline LLM02BM listed in Table 1 as determined at the 5% significancelevel when grown in the same environmental conditions.
 21. A method forproducing a hybrid corn seed comprising crossing a first inbred parentcorn plant with a second inbred parent corn plant and harvestingresultant hybrid corn seed, wherein the first inbred corn plant or thesecond inbred corn plant is the corn plant of claim
 9. 22. A method forproducing a hybrid corn seed, the method comprising: (a) planting inpollinating proximity a seed of the corn plant of claim 9 and a seed ofanother, different corn plant; (b) cultivating the seeds into plantsthat bear flowers; (c) allowing cross pollination to occur between thecultivated plants; and (d) harvesting seeds produced on at least one ofthe first inbred corn plant or the second inbred corn plant.
 23. Themethod according to claim 22, wherein the LLM02BM corn plant is the maleparent.
 24. The method according to claim 22, wherein the LLM02BM cornplant is the female parent.
 25. A hybrid corn seed produced by themethod according to claim
 22. 26. A hybrid corn plant, or parts thereof,produced by growing the hybrid corn seed of claim 25.